Mitogen-induced p53 downregulation precedes vascular smooth muscle cell migration from healthy tunica media and proliferation.

The tumor suppressor protein p53 plays an important role in the cell-cycle G(1) and G(2) checkpoints. In response to DNA damage, p53 can induce the transcription of p21, which inhibits the activation of various G(1) cyclin/cyclin-dependent kinase complexes. It is not known whether p53 plays a role in the initial migration of vascular smooth muscle cells from the arterial tunica media (mVSMCs). In this study, we have investigated whether mVSMC migration from healthy tunica media of young pigs and proliferation are regulated by p53. After 6 hours of incubation in mitogen-rich medium, explanted porcine tunica media tissue showed complete downregulation of p53 protein and p53 mRNA. The blockage of gene activity was not due to DNA methylation at the 5' control region of the gene. The mVSMC outgrowth did not show p53 expression. Mitogen-depletion of cultured p53(-)/mVSMCs did not restore p53 expression. Incubation of explanted porcine tunica media tissue in mitogen-deprived medium increased p53 protein content and blocked mVSMC outgrowth from the explant. As in p53-deficient rodent cells, mVSMCs incubated with colcemid overrode the spindle-dependent checkpoint, giving polyploidy and chromosomal pairing. UV-induced DNA damage in mVSMCs incubated with mitogen-free medium induced p53 expression and apoptotic cell death showing DNA nucleosomal laddering. However, UV-irradiated mVSMCs incubated in mitogen-rich medium did not express p53 and did not show cell death. In conclusion, our results demonstrate that early mVSMC migration from the tunica media requires mitogen-induced suppression of p53 that is highly expressed in contractile mVSMCs residing in the healthy vessel wall.

The effect of cyclic adenosine monophosphate on the mitogen-activated protein kinase pathway depends on both the cell type and the type of tyrosine kinase-receptor.

The mitogen-activated protein kinase (MAP kinase) is a key participant in growth factor-stimulated intracellular events such as proliferation and differentiation. We and others have previously described a cross-talk between the MAP kinase pathway and the cAMP pathway. Indeed, in several cell lines and, in particular in fibroblasts, an increase in the level of cAMP produced an inhibition of MAP kinase together with decreased cell proliferation. In contrast, in PC12 cells, cAMP induced an increase in the NGF-induced activation of MAP kinase concomitantly with augmented NGF-induced differentiation. Therefore, it has been proposed that the cellular context is important for the nature of the cAMP effects on growth factor-stimulated MAP kinase activity. Here we show that the type of tyrosine kinase receptor stimulated also participates in the nature of the cAMP effect. Thus, in NIH3T3 fibroblasts expressing NGF receptors (NIH3T3/trk cells) we found that cAMP potentiates NGF-stimulated ERK1 and MEK1 activities, whereas in NIH3T3 fibroblasts expressing insulin receptors (NIH3T3/IR cells) we saw no effect of cAMP on the activation of insulin-stimulated ERK1 and MEK1. In PC12 cells and in Rat1 fibroblasts expressing insulin receptors (PC12/IR and Rat1/IR cells) we observed, respectively, a potentiation and an inhibition of insulin-stimulated ERK1 activity. In addition, cAMP does not seem to modify the basal nor growth factor-stimulated She or IRS-1 tyrosine phosphorylation in the different cell lines studied. Finally, we observed that cAMP inhibited serum- and insulin-induced, but not NGF-induced, cell proliferation in NIH3T3 cells. However, cAMP potentiated insulin-stimulated cell differentiation in PC12/IR cells. These results led us to conclude that the cAMP effect on cell proliferation in NIH3T3 fibroblasts and PC12/IR cells appears to be correlated, in part, with the effect of cAMP on the MAP kinase pathway, but by itself this pathway cannot fully account for these observations.